Oxidative stress and decreased mitochondrial respiratory chain capacity are both implicated in normal aging and may contribute to the increased incidence of neurodegenerative disease. It is possible that the slow insidious onset of neurodegenerative disease is because neuronal damage may advance in a quantal manner, each downward step being initiated by an acute energy deficit. The aim of this research proposal is to investigate the consequences of such bioenergetic compromise upon the ability of primary neuronal cultures from mouse hippocampus and cerebellum to withstand the patho-physiological stresses of acute glutamate excitotoxicity and tetanic action potential firing. In order to model enhanced oxidative stress, cells will be cultured from mice heterozygous for the Sod2 gene encoding mitochondrial Mn-superoxide dismutase. Chronic complex I restriction will be modelled by culturing cells from the wild-type mice in the presence of low concentrations of the respiratory chain inhibitor rotenone. The central bioenergetic parameter to be monitored will be the mitochondrial membrane potential, membrane potential while the key cellular functions controlled by membrane potential namely ATP synthesis, Ca2+ sequestration and the generation and detoxification of reactive oxygen species (ROS), will be monitored in parallel. Oxidative stress derived from Ca2+loaded mitochondria is implicated in the excitotoxic cell death induced by chronic NMDA receptor activation. Since ROS generation by isolated mitochondria is highly dependent upon membrane potential the hypothesis will be investigated that endogenous or synthetic means of controlling membrane potential can alleviate the deleterious consequences to the neurons of these bioenergetic stresses. The role of the novel candidate uncoupling protein UCP5 in controlling membrane potential will be assessed by in vitro over-expression, while the efficacy of synthetic protophores and lipophilic cations will be investigated. The hypothesis that this project will test that fine control of membrane potential by endogenous or artificial means may be a mechanism to counteract any increase, resulting from age related mitochondrial function, in the susceptabilty of neurons to excititoxic or epileptiform stress. Bioenergetic, proteomic and genomic information will be integrated in this study. The long-term goals of this project are 1. To facilitate the design of more precise mouse models of aging. 2. To establish the role of mitochondria in normal aging and aging-related neurodegenrative disorder. 3. To indicate therapeutic strategies whereby the deleterious consequences of aging-related changes in mitochondrial function can be minimized.